Wild type HA-SV40 recombinant cloned at the unique Bam H1 site of pBR322 was used for the derivation of HA deletion mutants. A series of deletion mutations were introduced at a specific MboII site located 12 base pairs downstream of the initiation codon of HA. We identified one mutant with an in-phase deletion. The in-phase mutant HA sustained a deletion of 33 bp or 11 amino acids, all within the signal peptide. Sequence analysis predicted that 5 amino acids of the 16 amino-acid signal sequence remained as a result of this deletion. The signal peptide cleavage site of Gly-Gln and the subsequent downstream sequences were not affected. Mutant HA lacking the signal sequences accumulates in the cytoplasm but is not incorporated into the outer membrane. The mutant polypeptide synthesized during infection was not modified by glycosylation. This failure to agglutinate red blood cells suggests that the mutant HA remains monomeric with a resulting defect in forming trimeric structures which are required for functional activity. Alternatively, the functional defect of mutant HA may be due to the lack of carbohydrate components and as a result the mutant HA subunit fails to assemble properly.